The proposed studies deal with nuclear proteins and their assembly into macromolecular complexes with RNA on the chromosomes and in other nuclear organelles. Advantage will be taken of the giant nucleus and giant lampbrush chromosomes (LBCs) of the amphibian oocyte. Monoclonal antibodies will be produced against nuclear proteins, whose subnuclear distribution will be determined by immunofluorescence. The extraordinary morphological detail in LBCs allows antigen detection within single transcription units or in defined subnuclear particulates. The antibodies will be used to recover cDNA clones from expression libraries. Protein sequences derived from the cDNA clones will permit deductions about the role(s) of the proteins in nuclear function. In vitro transcriptions made from the cDNA clones will be injected into amphibian oocytes, and the translation products (polypeptides) will be followed biochemically and cytologically from their site of synthesis in the cytoplasm through the nuclear envelope to their final destination on the chromosomes or elsewhere in the nucleus. By appropriate in vitro alteration of the injected transcripts we will determine what amino acid sequences are necessary for transport across the nuclear envelope and for targeting to the final subnuclear destination. Studies will also be conducted on the transcription of the 5S gene sequences on LBCs by in situ nucleic acid hybridization. Finally studies will be carried out on a novel selfcleaving RNA transcribed from a short, repeated sequence of newt DNA, with emphasis on the possible existence of a ribonucleoprotein particle. All of the proposed studies deal with how RNA produced by the chromosomes becomes associated with nuclear proteins in order to carry out basic cellular functions (RNA processing, storage, and transport). Knowledge of such functions is fundamental for understanding cell metabolism in both the normal and diseased state.